An apparatus for analyzing blood samples in vitro typically requires fluids to be passed through a capillary tube in a predefined sequence, such as, air, isotonic fluids (ie., salt water), blood, isotonic fluids and then air. The apparatus typically includes one or more fluid-type detectors for determining the presence, or lack thereof, of blood within the capillary tube at various locations along the length of the tube within the instrument. Conventionally, the detection process is accomplished with an optical density measurement in which light of a known intensity is transmitted through the capillary tube and received and measured to determine the intensity of the light passing through the tube and to enable a determination of the density of the fluid within the tube. Since air and isotonic fluids are substantially clear in comparison to a blood sample, the presence of blood, as well as the leading and trailing edges of the blood sample within the sequence of fluids, is detected and identified by the optical density measurement.
Some blood processing apparatus are associated with an automated mechanism for generating blood smears on microscope slides. Such apparatus should measure, estimate, be supplied with, or otherwise determine the viscosity of a blood sample to determine smear hold time, velocity, and acceleration of a smear wedge component needed to create an optimal blood smear on the slide. Examples of automated slide making apparatus, smear wedge components, and related methods are disclosed in U.S. Pat. Nos. 5,650,332 and 5,804,145 issued to Gao et al. and U.S. Pat. No. 5,209,903 issued to Kanamori et al.
The movement of a blood-smearing member across the slide according to the Gao patents is controlled as a function of various predetermined physical parameters of the blood identified from blood analysis data. For instance, the primary parameter for determining smear wedge velocity is hematocrit HCT, although other hematology parameters are utilized to add or subtract to the velocity to correct for abnormal cases and/or for the presence of drugs. The Kanamori patent utilizes a pair of optical sensors and a timer to determine the amount of time required for a leading edge of the blood sample to pass from a first sensor to a second sensor located downstream thereof. The elapsed time measurement is utilized to determine the viscosity of the sample.
Some blood processing apparatus permit the erythrocyte sedimentation rate (ESR) and/or the zeta sedimentation rate (ZSR) of a blood sample to be determined. The ESR is a measure of the degree of settling of erythrocytes in plasma within an anticoagulated whole blood specimen during a period of time. The basic ESR measurement is the rate at which the turbid corpuscular part of the blood sample consisting of red and white blood cells and platelets separates from the nearly clear fluid plasma or serum. An elevated ESR is believed to be caused by an increase in the acute-phase asymmetrical proteins of plasma, largely fibrinogen, ∝2 globulin and γglobulin and is believed to indicate the presence of inflammation of the patient.
The ZSR is a measure of the packing of erythrocytes under a standardized stress (zetacrit). Integral proteins on red cell membranes contain sialic acid that provides erythrocytes with a negative charge. This negativity between cells, known as the so-called zeta potential, causes cells to repel one another as they move through the circulation system of the body. Altered plasma proteins, such as fibrinogen and globulins, in the surrounding medium, can cause a decrease in zeta potential. A decrease in zeta potential causes an increase in ESR. Thus, the ZSR measurement, expressed in % as the red cell hematocrit, assesses the ease with which the red blood cells pack under stress and is presumably related to the zeta potential of red blood cells when suspended in a particular plasma. A normal ZSR value for both males and females is in a range of 40% to 50% and is unaffected by anemia.
A method known as the Westergren method has been recommended by the International Council (formerly, Committee) for Standardization in Haematology as the method of choice for measuring ESR. This method has been utilized since the 1920s and is described in Br. J. Haematol., 24:671–673, 1973. Also see the following published references: Talkers, “Erythrocyte Sedimentation Rate/Zeta Sedimentation Rate”, Emer. Med. Clin. Of North America, Vol. 4, pp 87–93, February 1986; Moseley et al., “A Comparison of the Wintrobe, The Westergren and the ZSR Erthrocyte Sedimentation Rate (ESR) Methods to a Candidate Reference Method”, Clin Lab Haemat., Vol. 4, pp 169–178, 1982; and Bull et al., “The Zeta Sedimentation Ratio”, Blood, Vol. 40, pp 550–559, October 1972. The Westergren method is a gravity-based method in which a volume of blood is placed in a vertically oriented tube and in which the rate of sedimentation of the cells within the tube is recorded at fixed intervals over a period of time typically greater than an hour.
More recent examples of apparatus and methods for measuring sedimentation rates are disclosed by U.S. Pat. No. 3,848,796 issued to Bull and U.S. Pat. No. 5,827,746 issued to Duic. The Bull patent discloses a centrifuge apparatus, known as the so-called Zetafuge, that measures ZSR by applying a controlled centrifugation to a blood sample producing alternating compaction and dispersion of erythrocytes and by measuring how closely the erythrocytes approach one another under a specific standardized artificial gravitational force.
The Duic patent discloses an apparatus for measuring ESR in which a blood sample is preheated to an elevated temperature to minimize the viscosity of the sample and is then passed through a thin tube at a constant velocity in a manner that causes the cells to be densely packed within the center of the thin tube. Thereafter, the preheated blood sample is abruptly stopped thereby causing the plasma to stop. However, the kinetic energy and zeta potential of the cells cause the cells to continue moving forward and away from the center of the tube. A focused optical density measurement is performed through the center of the tube and an ESR measurement is obtained by recording a drop in optical attenuation based on the rate at which the cells move away from the center of the tube over a 30-second interval. This rate of particle movement within the sample is then extrapolated to the conventional gravity-based separation Westergren measurement.
Although the aforementioned apparatus, methods, systems and techniques may function satisfactorily for their intended purposes, the use of optical density measurements has some disadvantages and significant limitations. An optical density measurement can provide information only based on the average behavior of the fluid in the tube independent of cell velocities. An optical density measurement cannot be utilized to isolate the behavior of any one cross sectional position within a tube. For example, the optical density measurement cannot determine the peak velocity at the center of the tube nor can it differentiate the fluid velocity at the center of the tube relative to the fluid velocity at the edges of the tube. In addition, optical density sensors are only useful when used in combination with tubing have a small inner diameter that permits a sufficient amount of light to pass through the tubing and sample. Of course, use of small inner diameter tubing limits the fluid handling flow rate of samples through the hematology instrument.
The presence of microbubbles within a blood sample also presents a significant challenge since the presence of microbubbles are unrecognizable by optical density sensors and greatly effects the value of the optical density measurement. Further, optical density sensors must remain stationary relative to the tube through which the sensors obtain an optical density measurement for the sensors to remain properly calibrated. Any inadvertent movement of the tube relative to the sensors will require a time consuming recalibration of the sensors. Such inadvertent movement often occurs during the course of troubleshooting the blood-processing instrument for non-sensor related reasons.
A problem with generating blood smears is that hematology parametric data of blood samples lose value over time. Thus, a smear should be generated as quickly as possible after blood analysis and/or viscosity measurements to ensure that an optimal slide is created. Similarly, the measurement of ESR and ZSR typically require a significant initial dead time which has a significant effect on the time required for analysis. Such analysis, therefore, cannot readily be accomplished in succession with other analyses that can be performed much quicker, such as, for instance blood cell counts.
The ESR measurement method and apparatus according to U.S. Pat. No. 5,827,746 issued to Duic also has disadvantages due to its reliance on optical density measurements. An ESR measurement is dependent on the temperature and viscosity of the sample, the protein concentration in the plasma, the erythrocyte size bias, and lipids. Lipids cause problems related to carryover and baseline drift of the optical density measurement. When a fluid column is brought to a stop, lipids will electrostatically be attracted to walls of the tubing and will continue to build-up on the walls. Platelets can also become attached to the tubing walls. The build-up causes an increase in optical attenuation of the tube and therefore, affects the optical density measurement. Thus, there is an unknown progressive error build-up that distorts the optical density and ESR measurements of all samples processed sequentially through the apparatus. The optical density measurement is also effected when a portion of the build-up is torn loose from the walls of the tube and flows within the blood sample being analyzed. Another limitation of the above stated method is that it requires an additional time-consuming process step of preheating the blood sample to a precise elevated temperature before the test can be started.
Abnormal blood samples (ie., samples with elevated ESR which are most important to identify) often have extreme blood cell counts and plasma viscosity that cause the cells to dissipate more slowly in the fluid column. Blood samples may alternatively have a low viscosity and low cell count resulting in the cells dissipating at a faster than normal rate. Neither of these conditions can be recognized, determined and/or corrected for when an optical density measurement provides the mechanism for measuring ESR.
With the foregoing in mind, a primary object of the present invention is to provide an improved apparatus and method for analyzing a liquid located in a capillary tube of a hematology instrument.
Another object of the present invention is to provide an apparatus and method that can accurately, readily and quickly determine the density of a liquid contained in a tube of a hematology instrument and whether or not microbubbles are present within the liquid.
A further object of the present invention is to provide an apparatus and method that can accurately, readily and quickly determine the velocity of individual cells within a blood sample flowing through a capillary tube and the viscosity of the blood sample.
A still further object of the present invention is to provide an apparatus and method that can accurately, readily and quickly determine the ESR and ZSR of a blood sample.
Yet another object of the present invention is to provide apparatus that is capable of use in daily operations in a cost efficient manner at relatively high fluid handling flow rates requiring only a minimum of skill to operate, utilize and maintain.